Mating system parameters at hierarchical levels of fruits, individuals and populations in the Brazilian insect-pollinated tropical tree, Tabebuia roseo-alba (Bignoniaceae)

For many tree species, mating system analyses have indicated potential variations in the selfing rate and paternity correlation among fruits within individuals, among individuals within populations, among populations, and from one flowering event to another. In this study, we used eight microsatellite markers to investigate mating systems at two hierarchical levels (fruits within individuals and individuals within populations) for the insect pollinated Neotropical tree Tabebuia roseo-alba. We found that T. roseo-alba has a mixed mating system with predominantly outcrossed mating. The outcrossing rates at the population level were similar across two T. roseo-alba populations; however, the rates varied considerably among individuals within populations. The correlated paternity results at different hierarchical levels showed that there is a high probability of shared paternal parentage when comparing seeds within fruits and among fruits within plants and full-sibs occur in much higher proportion within fruits than among fruits. Significant levels of fixation index were found in both populations and biparental inbreeding is believed to be the main cause of the observed inbreeding. The number of pollen donors contributing to mating was low. Furthermore, open-pollinated seeds varied according to relatedness, including half-sibs, full-sibs, self-sibs and self- half-sibs. In both populations, the effective population size within a family (seed-tree and its offspring) was lower than expected for panmictic populations. Thus, seeds for ex situ conservation genetics, progeny tests and reforestation must be collected from a large number of seed-trees to guarantee an adequate effective population in the sample.

Reproductive biology and species geographical distribution in the Melastomataceae: a survey based on New World taxa

Apomictic plants are less dependent on pollinator services and able to occupy more diverse habitats than sexual species. However, such assumptions are based on temperate species, and comparable evaluation for species-rich Neotropical taxa is lacking. In this context, the Melastomataceae is a predominantly Neotropical angiosperm family with many apomictic species, which is common in the Campos Rupestres, endemism-rich vegetation on rocky outcrops in central Brazil. In this study, the breeding system of some Campo Rupestre Melastomataceae was evaluated, and breeding system studies for New World species were surveyed to test the hypothesis that apomixis is associated with wide distributions, whilst sexual species have more restricted areas. The breeding systems of 20 Campo Rupestre Melastomataceae were studied using hand pollinations and pollen-tube growth analysis. In addition, breeding system information was compiled for 124 New World species of Melastomataceae with either wide (1000 km) or restricted distributions. Most (80 ) of the Campo Rupestre species studied were self-compatible. Self-incompatibility in Microlicia viminalis was associated with pollen-tube arrest in the style, as described for other Melastomataceae, but most self-incompatible species analysed showed pollen-tube growth to the ovary irrespective of pollination treatment. Apomictic species showed lower pollen viability and were less frequent among the Campo Rupestre plants. Among the New World species compiled, 43 were apomictic and 77 sexual (24 self-incompatible and 53 self-compatible). Most apomictic (86 ) and self-incompatible species (71 ) presented wide distributions, whilst restricted distributions predominate only among the self-compatible ones (53 ). Self-compatibility and dependence on biotic pollination were characteristic of Campo Rupestre and narrowly distributed New World Melastomataceae species, whilst apomictics are widely distributed. This is, to a certain extent, similar to the geographical parthenogenesis pattern of temperate apomictics.

Mouse Housing System Using Pressurized Cages Intraventilated by Direct-Current Microfans

We performed the initial assessment of an alternative pressurized intraventilated (PIV) caging system for laboratory mice that uses direct-current microfans to achieve cage pressurization and ventilation. Twenty-nine pairs of female SPF BALB/c mice were used, with 19 experimental pairs kept in Ply cages and 10 control pairs kept in regular filter-top (FT) cages. Both groups were housed in a standard housing room with a conventional atmospheric control system. For both systems, intracage temperatures were in equilibrium with ambient room temperature. PIV cages showed a significant difference in pressure between days 1 and 8. Air speed (and consequently airflow rate) and the number of air changes hourly in the PIV cages showed decreasing trends. In both systems, ammonia concentrations increased with time, with significant differences between groups starting on day 1. Overall, the data revealed that intracage pressurization and ventilation by using microfans is a simple, reliable system, with low cost, maintenance requirements, and incidence of failures. Further experiments are needed to determine the potential influence of this system on the reproductive performance and pulmonary integrity in mice.

The role of AT1 receptor-mediated reproductive function in renovascular hypertension in male rats

There is an association between hypertension and reproductive dysfunction. Angiotensin II (Ang II) is involved in the pathogenesis of hypertension and the regulation of reproduction. The present study aimed to determine whether the angiotensinergic system mediates the effects of hypertension on ieproductive function in male rats subjected to a two-kidney, one-clip (2K1C) model. Sexual behavior parameters, gametogenesis and plasma concentrations of Ang II, testosterone, prolactin and corticosterone were evaluated in male rats 28 days after 2K1C or sham surgery and losartan (Los) treatment (a type 1 angiotensin II (All) receptor antagonist) or vehicle (V) treatment. The animals were divided into Sham + V, 2K1C + V. Sham + Los and 2K1C + Los groups. The 2KiC + V group showed a hypertensive response, inhibition of sexual behavior, spermatogenesis dysfunction, and increases in plasma Ang II and prolactin. Conversely, plasma testosterone decreased, and plasma corticosterone remained constant. Losartan treatment normalized blood pressure and prevented the changes in plasma testosterone and prolactin, sexual behavior and spermatogenesis in the 2KiC + Los group. In addition, losartan treatment caused an additional increase in circulating Ang II in both groups (She m + Los arid 2K1C + Los). Together, these results suggest that Ang II, acting through the All receptor, modulates behavioral and endocrine parameters of reproductive function during renovascular hypertension. In addition, the effects of circulating Ang II on plasma testosterone and prolactin seem to contribute to the spermatogenic and sexual dysfunctions in hypertensive rats. (C) 2012 Els.evier Inc. All rights reserved.

Prenatal and Neonatal Adaptations with a Focus on the Respiratory System

Contents Among the modifications that occur during the neonatal period, pulmonary development is the most critical. The neonate's lungs must be able to perform adequate gas exchange, which was previously accomplished by the placenta. Neonatal respiratory distress syndrome is defined as insufficient surfactant production or pulmonary structural immaturity and is specifically relevant to preterm newborns. Prenatal maternal betamethasone treatment of bitches at 55days of gestation leads to structural changes in the neonatal lung parenchyma and consequently an improvement in the preterm neonatal respiratory condition, but not to an increase in pulmonary surfactant production. Parturition represents an important challenge to neonatal adaptation, as the uterine and abdominal contractions during labour provoke intermittent hypoxia. Immediately after birth, puppies present venous mixed acidosis (low blood pH and high dioxide carbon saturation) and low but satisfactory Apgar scores. Thus, the combination of physiological hypoxia during birth and the initial effort of filling the pulmonary alveoli with oxygen results in anaerobiosis. As a neonatal adaptation follow-up, the Apgar analysis indicates a tachypnoea response after 1h of life, which leads to a shift in the blood acidbase status to metabolic acidosis. One hour is sufficient for canine neonates to achieve an ideal Apgar score; however, a haemogasometric imbalance persists. Dystocia promotes a long-lasting bradycardia effect, slows down Apgar score progression and aggravates metabolic acidosis and stress. The latest data reinforce the need to accurately intervene during canine parturition and offer adequate medical treatment to puppies that underwent a pathological labour.

Immunohistochemical study of pituitary cells in wild and captive Salminus hilarii (Characiformes: Characidae) females during the annual reproductive cycle

Freshwater fish that live exclusively in rivers are at particular risk from fragmentation of the aquatic system, mainly the species that migrate upriver for reproduction. That is the case of Salminus hilarii, an important migratory species currently classified as “almost threatened” in the São Paulo State (Brazil), facing water pollution, dam construction, riparian habitat destruction and environmental changes that are even more serious in this State. Additionally, this species show ovulation dysfunction in captivity. Our studies focused on the identification and distribution of the pituitary cell types in the adenohypophysis of S. hilarii females, including a morphometric analysis that compares pituitary cells from wild and captive broodstocks during the reproductive annual cycle. The morphology of adenohypophysial cells showed differences following the reproductive cycle and the environment. In general, optical density suggested a higher cellular activity during the previtellogenic (growth hormone) and vitellogenic (somatolactin) stages in both environments. Additionally, the nucleus/cell ratio analysis suggested that growth hormone and somatolactin cells were larger in wild than in captive females in most reproductive stages of the annual cycle. In contrast, prolactin hormone showed no variation throughout the reproductive cycle (in both environments). Morphometrical analyses related to reproduction of S. hilarii in different environmental conditions, suggest that somatolactin and growth hormone play an important role in reproduction in teleost and can be responsible for the regulation of associated processes that indirectly affect reproductive status.

Immunohistochemical study of pituitary cells in wild and captive Salminus hilarii (Characiformes: Characidae) females during the annual reproductive cycle

Freshwater fish that live exclusively in rivers are at particular risk from fragmentation of the aquatic system, mainly the species that migrate upriver for reproduction. That is the case of Salminus hilarii, an important migratory species currently classified as “almost threatened” in the São Paulo State (Brazil), facing water pollution, dam construction, riparian habitat destruction and environmental changes that are even more serious in this State. Additionally, this species show ovulation dysfunction in captivity. Our studies focused on the identification and distribution of the pituitary cell types in the adenohypophysis of S. hilarii females, including a morphometric analysis that compares pituitary cells from wild and captive broodstocks during the reproductive annual cycle. The morphology of adenohypophysial cells showed differences following the reproductive cycle and the environment. In general, optical density suggested a higher cellular activity during the previtellogenic (growth hormone) and vitellogenic (somatolactin) stages in both environments. Additionally, the nucleus/cell ratio analysis suggested that growth hormone and somatolactin cells were larger in wild than in captive females in most reproductive stages of the annual cycle. In contrast, prolactin hormone showed no variation throughout the reproductive cycle (in both environments). Morphometrical analyses related to reproduction of S. hilarii in different environmental conditions, suggest that somatolactin and growth hormone play an important role in reproduction in teleost and can be responsible for the regulation of associated processes that indirectly affect reproductive status.

Use of E. coli OMVs as delivery system to elicit neutralizing antibodies against Chlamydia infectivity: the HtrA protein example

The obligate intracellular pathogen Chlamydia trachomatis is a gram negative bacterium which infects epithelial cells of the reproductive tract. C. trachomatis is the leading cause of bacterial sexually transmitted disease worldwide and a vaccine against this pathogen is highly needed. Many evidences suggest that both antigen specific-Th1 cells and antibodies may be important to provide protection against Chlamydia infection. In a previous study we have identified eight new Chlamydia antigens inducing CD4-Th1 and/or antibody responses that, when combined properly, can protect mice from Chlamydia infection. However, all selected recombinant antigens, upon immunization in mice, elicited antibodies not able to neutralize Chlamydia infectivity in vitro. With the aim to improve the quality of the immune response by inducing effective neutralizing antibodies, we used a novel delivery system based on the unique capacity of E. coli Outer Membrane Vesicles (OMV) to present membrane proteins in their natural composition and conformation. We have expressed Chlamydia antigens, previously identified as vaccine candidates, in the OMV system. Among all OMV preparations, the one expressing HtrA Chlamydia antigen (OMV-HtrA), showed to be the best in terms of yield and quantity of expressed protein, was used to produce mice immune sera to be tested in neutralization assay in vitro. We observed that OMV-HtrA elicited specific antibodies able to neutralize efficiently Chlamydia infection in vitro, indicating that the presentation of the antigens in their natural conformation is crucial to induce an effective immune response. This is one of the first examples in which antibodies directed against a new Chlamydia antigen, other than MOMP (the only so far known antigen inducing neutralizing antibodies), are able to block the Chlamydia infectivity in vitro. Finally, by performing an epitope mapping study, we investigated the specificity of the antibody response induced by the recombinant HtrA and by OMV-HtrA. In particular, we identified some linear epitopes exclusively recognized by antibodies raised with the OMV-HtrA system, detecting in this manner the antigen regions likely responsible of the neutralizing effect.

Pollination ecology and reproductive success in isolated populations of flowering plants: Primula apennina Widmer, Dictamnus albus L. and Convolvulus lineatus L.

This research focuses on reproductive biology and pollination ecology of entomophilous angiosperms, with particular concern to reproductive success in small and isolated populations of species that occur at their distribution limits or are endemic. I considered three perennial herbs as model species: Primula apennina Widmer, Dictamnus albus L. and Convolvulus lineatus L. I carried out field work on natural populations and performed laboratory analyses on specific critical aspects (resource allocation, pollen viability, stigmatic receptivity, physiological self-incompatibility, seed viability), through which I analysed different aspects related to plant fitness, such as production of viable seed, demographic structure of populations, type and efficiency of plant-pollinator system, and limiting factors.

Taxonomy, phylogeny and reproductive ecology of Gentiana lutea L.

This research focuses on taxonomy, phylogeny and reproductive ecology of Gentiana lutea. L.. Taxonomic analysis is a critical step in botanical studies, as it is necessary to recognize taxonomical unit. Herbarium specimens were observed to assess the reliability of several subspecies-diagnostic characters. The analysis of G. lutea genetic variability and the comparison with that of the other species of sect. Gentiana were performed to elucidate phylogenetic relationships among G. lutea subspecies and to propose a phylogenetic hypothesis for the evolution and the colonization dynamics of the section. Appropriate scientific information is critical for the assessment of species conservation status and for effective management plans. I carried out field work on five natural populations and performed laboratory analyses on specific critical aspects, with special regard to G. lutea breeding system and type and efficiency of plant-pollinator system. Bracts length is a reliable character to identify subsp. vardjanii, however it is not exclusive, hence to clearly identify subsp. vardjanii, other traits have to be considered. The phylogenetic hypotheses obtained from nuclear and chloroplast data are not congruent. Nuclear markers show a monophyly of sect. Gentiana, a strongly species identity of G. lutea and clear genetic identity of subsp. vardjanii. The little information emerging from plastid markers indicate a weak signal of hybridization and incomplete sorting of ancestral lineages. G. lutea shows a striking variation in intra-floral dichogamy probably evolved to reduce pollen-stigma interference. Although the species is partially self-compatible, pollen vectors are necessary for a successful reproduction, and moreover it shows a strong inbreeding depression. G. lutea is a generalist species: within its spectrum of visitors is possible to recognize "nectar thieves" and pollinators with sedentary or dynamic behaviour. Pollen limitation is frequent and it could be mainly explained by poor pollen quality.

Alternative reproductive tactics, sexual selection and the effects of inbreeding in the ant Hypoponera opacior

Inbreeding can lead to a fitness reduction due to the unmasking of deleterious recessive alleles and the loss of heterosis. Therefore, most sexually reproducing organisms avoid inbreeding, often by disperal. Besides the avoidance of inbreeding, dispersal lowers intraspecific competition on a local scale and leads to a spreading of genotypes into new habitats. In social insects, winged reproductives disperse and mate during nuptial flights. Therafter, queens independently found a new colony. However, some species also produce wingless sexuals as an alternative reproductive tactic. Wingless sexuals mate within or close to their colony and queens either stay in the nest or they found a new colony by budding. During this dependent colony foundation, wingless queens are accompanied by a fraction of nestmate workers. The production of wingless reproductives therefore circumvents the risks associated with dispersal and independent colony foundation. However, the absence of dispersal can lead to inbreeding and local competition.rnIn my PhD-project, I investigated the mating biology of Hypoponera opacior, an ant that produces winged and wingless reproductives in a population in Arizona. Besides the investigation of the annual reproductive cycle, I particularly focused on the consequences of wingless reproduction. An analysis of sex ratios in wingless sexuals should reveal the relative importance of local resource competition among queens (that mainly compete for the help of workers) and local mate competition among males. Further, sexual selection was expected to act on wingless males that were previously found to mate with and mate-guard pupal queens in response to local mate competition. We studied whether males are able to adapt their mating behaviour to the current competitive situation in the nest and which traits are under selection in this mating situation. Last, we investigated the extent and effects of inbreeding. As the species appeared to produce non-dispersive males and queens quite frequently, we assumed to find no or only weak negative effects of inbreeding and potentially mechanisms that moderate inbreeding levels despite frequent nest-matings.rnWe found that winged and wingless males and queens are produced during two separate seasons of the year. Winged sexuals emerge in early summer and conduct nuptial flights in July, when climate conditions due to frequent rainfalls lower the risks of dispersal and independent colony foundation. In fall, wingless sexuals are produced that reproduce within the colonies leading to an expansion on the local scale. The absence of dispersal during this second reproductive season resulted in a local genetic population viscosity and high levels of inbreeding within the colonies. Male-biased sex ratios in fall indicated a greater importance of local resource competition among queens than local mate competition among males. Males were observed to adjust mate-guarding durations to the competitive situation (i.e. the number of competing males and pupae) in the nest, an adaptation that helps maximising their reproductive success. Further, sexual selection was found to act on the timing of emergence as well as on body size in these males, i.e. earlier emerging and larger males show a higher mating success. Genetic analyses revealed that wingless males do not actively avoid inbreeding by choosing less related queens as mating partners. Further, we detected diploid males, a male type that is produced instead of diploid females if close relatives mate. In contrast to many other Hymenopteran species, diploid males were here viable and able to sire sterile triploid offspring. They did not differ in lifespan, body size and mating success from “normal” haploid males. Hence, diploid male production in H. opacior is less costly than in other social Hymenopteran species. No evidence of inbreeding depression was found on the colony level but more inbred colonies invested more resources into the production of sexuals. This effect was more pronounced in the dispersive summer generation. The increased investment in outbreeding sexuals can be regarded as an active strategy to moderate the extent and effects of inbreeding. rnIn summary, my thesis describes an ant species that has evolved alternative reproductive tactics as an adaptation to seasonal environmental variations. Hereby, the species is able to maintain its adaptive mating system without suffering from negative effects due to the absence of dispersal flights in fall.rn

Controlled reproduction in Anguilla anguilla (L.): advanced studies on broodstock management, spawning techniques and system design for artificial seed production.

The thesis aims to expose the advances achieved in the practices of captive breeding of the European eel (Anguilla anguilla). Aspects investigated concern both approaches livestock (breeding selection, response to hormonal stimulation, reproductive performance, incubation of eggs) and physiological aspects (endocrine plasma profiles of players), as well as engineering aspects. Studies conducted on various populations of wild eel have shown that the main determining factor in the selection of wild females destined to captive breeding must be the Silver Index which may determine the stage of pubertal development. The hormonal induction protocol adopted, with increasing doses of carp pituitary extract, it has proven useful to ovarian development, with a synchronization effect that is positively reflected on egg production. The studies on the effects of photoperiod show how the condition of total darkness can positively influence practices of reproductions in captivity. The effects of photoperiod were also investigated at the physiological level, observing the plasma levels of steroids ( E2, T) and thyroid hormones (T3 and T4) and the expression in the liver of vitellogenin (vtg1 and vtg2) and estradiol membrane receptor (ESR1). From the comparison between spontaneous deposition and insemination techniques through the stripping is inferred as the first ports to a better qualitative and quantitative yield in the production of eggs capable of being fertilized, also the presence of a percentage of oocytes completely transparent can be used to obtain eggs at a good rate of fertility. Finally, the design and implementation of a system for recirculating aquaculture suited to meet the needs of species-specific eel showed how to improve the reproductive results, it would be preferable to adopt low-flow and low density incubation.

Male-biased reproductive effort in a long-lived seabird.

Background: In dimorphic seabirds, the larger sex tends to provision more than the smaller sex. In contrast, monogamy and biparental care are often associated with equal effort between the sexes. However, the few studies that have tested sex-specific effort in monomorphic seabirds have primarily examined the details of foraging at sea. Hypotheses: Parental effort is also sex-biased in a monomorphic seabird mating system for one of two reasons: (1) If females enter the period of parental care less able to invest in care due to the cost of egg production, male-biased effort may be necessary to avoid reproductive failure. (2) Alternatively, female-biased effort may occur due to the initial disparity in gamete size, particularly in species with internal fertilization. Organism: Leach’s storm-petrel (Oceanodroma leucorhoa), a monomorphic seabird with true monogamy and obligate biparental care. Site: A breeding colony of Oceanodroma leucorhoa at the Bowdoin Scientific Station on Kent Island, Bay of Fundy, New Brunswick, Canada. Methods: Across multiple breeding seasons, we assessed incubation behaviour and chickrearing behaviour through one manipulative and multiple observational studies. We assessed energetic investment by inducing feather replacement and measuring the resulting rate of feather growth during both the incubation and chick-rearing phases of parental care. Conclusions: We observed male-biased effort. Males incubated the egg for a greater proportion of time than did females and, when faced with an egg that would not hatch, males continued to incubate past the point when females abandoned it. Males made a higher percentage of total food deliveries to chicks than did females, resulting in greater mean daily food provisioning by males than by females. During chick rearing, males grew replacement feathers more slowly than did females, indicating that males were more likely to reduce their own nutritional condition while raising chicks than were females. These results support the hypothesis that females enter the period of parental care at a nutritional deficit and males must compensate to avoid reproductive failure.

Host condition and host immunity affect parasite fitness in a bird-ectoparasite system

1. Parasites might preferentially feed on hosts in good nutritional condition as such hosts provide better resources for the parasites' own growth, survival and reproduction. However, hosts in prime condition are also better able to develop costly immunological or physiological defence mechanisms, which in turn reduce the parasites' reproductive success. The interplay between host condition, host defence and parasite fitness will thus play an important part in the dynamics of host-parasite systems.;2. In a 2 x 2 design, we manipulated both the access to food in great tit Parus major broods and the exposure of the nestlings to hen fleas Ceratophyllus gallinae, a common ectoparasite of hole-breeding birds. We subsequently investigated the role of manipulated host condition, host immunocompetence, and experimentally induced host defence in nestlings on the reproductive success of individual hen flea females.;3. The food supplementation of the nestlings significantly influenced the parasites' reproductive success. Female fleas laid significantly more eggs when feeding on food-supplemented hosts.;4. Previous parasite exposure of the birds affected the reproductive success of fleas. However, the impact of this induced host response on flea reproduction depended on the birds' natural level of immunocompetence, assessed by the phytohaemagglutinin (PHA) skin test. Flea fecundity significantly decreased with increasing PHA response of the nestlings in previously parasite-exposed broods. No relationship between flea fitness and host immunocompetence was, however, found in previously unexposed broods. The PHA response thus correlates with the nestlings' ability to mount immunological or physiological defence mechanisms against hen fleas. No significant interaction effect between early flea exposure and food supplementation on the parasites' reproductive success was found.;5. Our study shows that the reproductive success of hen fleas is linked to the hosts' food supply early in life and their ability to mount induced immunological or physiological defence mechanisms. These interactions between host quality and parasite fitness are likely to influence host preference, host choice and parasite virulence and thus the evolutionary dynamics in host-parasite systems.

The Accumulation of Reproductive Incompatibilities in African Cichlid Fish

The rate at which different components of reproductive isolation accumulate with divergence time between species has only been studied in a limited, but growing, number of species. We measured premating isolation and hybrid inviability at four different ontogenetic stages from zygotes to adults in interspecific hybrids of 26 pairs of African cichlid species, spanning the entire East African haplochromine radiation. We then used multiple relaxed molecular clock calibrations to translate genetic distances into absolute ages to compare evolutionary rates of different components of reproductive isolation. We find that premating isolation accumulates fast initially but then changes little with increasing genetic distance between species. In contrast, postmating isolation between closely related species is negligible but then accumulates rapidly, resulting in complete hybrid inviability after 4.4/8.5/18.4 million years (my). Thus, the rate at which complete intrinsic incompatibilities arise in this system is orders of magnitude lower than rates of speciation within individual lake radiations. Together these results suggest divergent ecological adaptations may prevent populations from interbreeding and help maintain cichlid species diversity, which may be vulnerable to environmental degradation. By quantifying the capacity to produce viable hybrids between allopatric, distantly related lineages our results also provide an upper divergence time limit for the "hybrid swarm origin" model of adaptive radiation.